The goal of this project is to determine if decreased expression of ATM, a well characterized tumor suppressor important in the maintenance of genome integrity, occurs in breast cancer. Epidemiologic studies on ATM heterozygotes determined that these individuals display an elevated risk for breast cancer, presumably due to decreased ATM abundance. However, whether reduced ATM expression is a factor in sporadic breast cancer is currently unresolved. We have recently shown that aberrant methylation of CpG dinucleotides within the proximal promoter region of the ATM gene gives rise to dramatic downregulation of ATM expression and phenotypic consequences associated with reduced ATM abundance (e.g., increased radiosensitivity). Aberrant promoter methylation, a phenomenon termed epigenetic silencing, leads to decreased expression of numerous tumor suppressors in breast and other cancers. Based on our findings, we hypothesize that ATM is a novel target for epigenetic silencing in breast cancer. We will examine this hypothesis by addressing the following aims: 1) We will test our hypothesis that breast cancer cell lines exhibit diminished ATM expression due to aberrant ATM promoter methylation. This hypothesis will be examined by determining: a) relative ATM protein abundance in an extensive panel of cultured breast tumor lines by immunoblotting; b) relative ATM mRNA abundance in cell lines displaying reduced ATM protein expression by quantitative, real-time PCR; c) methylation status of the ATM promoter in cell lines showing reduced ATM protein and mRNA expression using a variety of approaches to that score methylation in genomic DNA. 2) We will test our hypothesis that surgically obtained breast adenocarcinomas exhibit diminished ATM expression due to aberrant ATM promoter methylation. We will test this hypothesis by determining: a) relative ATM mRNA abundance in breast tumors by real-time PCR; b) methylation of status of the ATM promoter in malignant cells showing reduced ATM expression. Completion of this research program will result in a greater understanding of the occurrence of decreased ATM expression in breast cancer, and whether this event can be used as a prognostic marker in this disease. Because reduced ATM expression renders cells more sensitive to the cytotoxic effects of ionizing radiation, these studies could prove useful in predicting the effectiveness of radiotherapy.